Figure 5

Western analysis of stable transfected cell lines expressing mutant BLM alleles. A. Whole cell lysates (15 μg total protein) from uninduced and induced cells were resolved on 4-12% SDS polyacrylamide gels and transferred to PVDF membranes. The bound complexes were detected with ECL reagents and exposed to x-ray film: 1) GFP-BLM+Tet, 2) GFP-ΔN1 no Tet, 3) GFP-ΔN1+Tet, 4) GFP-ΔN2 no Tet, 5) GFP-ΔN2+Tet. 6) GFP-ΔN3 no Tet, 7) GFP-ΔN3+Tet, 8) GFP-ΔN4 no Tet, 9) GFP-ΔN4+Tet. B. ECL western analysis of whole cell lysates: 1) GFP-BLM+Tet, 2) GFP-K695T no Tet, 3) GFP-K695T+Tet, 4) GFP-ΔH1 no Tet, 5) GFP-ΔH1+Tet, 6) GFP-ΔC1 no Tet, 7) GFP-ΔC1+Tet, 8) GFP-ΔC2 no Tet, 9) GFP-ΔC2+Tet. The positions of molecular weight standards are indicated to the left. Filters were reacted with α-GFP and goat α-mouse secondary conjugated with horseradish peroxidase. The bound complexes were detected with ECL reagents and exposure to x-ray film. C. Identical filters were reacted with α-GFP and goat α-mouse secondary conjugated with alkaline phosphatase. The fluorescent ECF product was visualized with a Molecular Dynamics Storm. The fluorescent image was quantitated using Imagequant. The values were normalized to normal GFP-BLM expression.